Anti-sickling hemoglobin

ABSTRACT

Disclosed are anti-sickling human hemoglobins for use as sickle cell anemia therapeutics.

This application is a continuation-in-part of U.S. Ser. No. 08/080,664,filed Jun. 21, 1993, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to recombinant anti-sickling hemoglobins suitablefor use as therapeutics for the treatment of sickle cell anemia.

The gene that encodes hemoglobin S (the defect leading to sickle cellanemia) is inherited as an autosomal trait and occurs in theheterozygous condition as the sickle trait in 8-10% of black persons inthe Unites States. Two major clinical features characterize sickle cellanemia: (1) chronic hemolysis that is stable and only moderatelydebilitating, and (2) acute, episodic vaso-occlusive crises that causeorgan failure and account for most of the mortality and morbidityassociated with the disease.

The molecular basis for sickle cell disease is an A to T transversion inthe 6th codon of the human β-globin gene. This simple transversionchanges a polar glutamic acid residue to a non-polar valine (Ingram etal., Nature 178:792, 1956; Ingram et al., Nature 180:326, 1957) in theβ-globin polypeptide and, thus, drastically decreases the solubility ofthis hemoglobin (termed Hb S). When the intracellular concentration ofHb S is high and the partial pressure of oxygen is low in the capillarybeds, the non-polar valine, which is on the surface of the hemoglobinmolecule, interacts with two other non-polar residues on the surface ofa second hemoglobin molecule, and initiates aggregation (Padlan et al.,J. Biol. Chem. 260:8280-8291, 1985; Wishner et al., J. Mol. Biol.98:179-194, 1975). Once approximately 10 hemoglobin monomers interact,long polymers rapidly accumulate, and complex 14-stranded fibers areformed (Crepeau et al., Nature 274:616-617, 1978; Dykes et al., J. Mol.Biol. 130:451-472, 1979; Eaton et al., Blood 70:1245-1266, 1987;Hofrichter et al., Proc. Natl. Acad. Sci USA 71:4864-4868, 1974). Theformation of these fibers reduces the flexibility of red blood cells andleads to the occlusion of small capillaries. Intracellular fiberformation also results in erythrocyte membrane damage and increased redcell lysis (Noguchi et al., Blood 58:1057, 1981; Brittenham et al.,Blood 65:183, 1985). The ensuing disease is characterized by a chronichemolytic anemia with episodes of severe pain, and tissue damage thatcan result in stroke, kidney failure, heart disease, infection, andother complications (Bunn et al., Hemoglobin: Molecular, Genetic, andClinical Aspects. (W. B. Saunders, Philadelphia, 1986)).

SUMMARY OF THE INVENTION

In one aspect, the invention features recombinant human hemoglobin withanti-sickling activity. Preferably, such anti-sickling hemoglobin isderived from β-globin. In preferred embodiments, the anti-sicklinghemoglobin includes a mutation which disrupts the hydrophobic pocketformed by β-globin amino acids phenylalanine 85 and leucine 88, butwhich leaves intact the correct positioning of the heme moiety.Preferred anti-sickling human hemoglobins include: (a) a glutamineresidue at β-globin amino acid 87; (b) a lysine residue at β-globinamino acid 87; (c) a lysine residue at β-globin amino acid 80; (d) analanine residue at β-globin amino acid 22; (e) a glutamine residue atβ-globin amino acid 87 and an alanine residue at β-globin amino acid 22;(f) a lysine residue at β-globin amino acid 87 and an alanine residue atβ-globin amino acid 22; (g) a lysine residue at β-globin amino acid 80and an alanine residue at β-globin amino acid 22; (h) a lysine residueat β-globin amino acid 108; (i) a lysine residue at β-globin amino acid108 and an alanine residue at β-globin amino acid 22; (j) a lysineresidue at β-globin amino acid 108 and a glutamine residue at β-globinamino acid 87; (k) a lysine residue at β-globin amino acid 108 and alysine residue at β-globin amino acid 87; (1) a lysine residue atβ-globin amino acid 108 and a lysine residue at β-globin amino acid 80;(m) a lysine residue at β-globin amino acid 108, an alanine residue atβ-globin amino acid 22, and a glutamine residue at β-globin amino acid87; (n) a lysine residue at β-globin amino acid 108, an alanine residueat β-globin amino acid 22, and a lysine residue at β-globin amino acid87; (o) a lysine residue at β-globin amino acid 108, an alanine residueat β-globin amino acid 22, and a lysine residue at β-globin amino acid80; (p) a glutamic acid residue at β-globin amino acid 95; (q) aglutamic acid residue at β-globin amino acid 95 and an alanine residueat β-globin amino acid 22; (r) a glutamic acid residue at β-globin aminoacid 95 and a glutamine residue at β-globin amino acid 87; (s) aglutamic acid residue at β-globin amino acid 95 and a lysine residue atβ-globin amino acid 87; (t) a glutamic acid residue at β-globin aminoacid 95 and a lysine residue at β-globin amino acid 80; (u) a glutamicacid residue at β-globin amino acid 95, an alanine residue at β-globinamino acid 22, and a glutamine residue at β-globin amino acid 87; (v) aglutamic acid residue at β-globin amino acid 95, an alanine residue atβ-globin amino acid 22, and a lysine residue at β-globin amino acid 87;(w) a glutamic acid residue at β-globin amino acid 95, an alanineresidue at β-globin amino acid 22, and a lysine residue at β-globinamino acid 80; (x) an aspartic acid residue at β-globin amino acid 16;(y) an aspartic acid residue at β-globin amino acid 16 and an alanineresidue at β-globin amino acid 22; (z) an aspartic acid residue atβ-globin amino acid 16 and a glutamine residue at β-globin amino acid87; (a') an aspartic acid residue at β-globin amino acid 16 and a lysineresidue at β-globin amino acid 87; (b') an aspartic acid residue atβ-globin amino acid 16 and a lysine residue at β-globin amino acid 80;(c') an aspartic acid residue at β-globin amino acid 16, an alanineresidue at β-globin amino acid 22, and a glutamine residue at β-globinamino acid 87; (d') an aspartic acid residue at β-globin amino acid 16,an alanine residue at β-globin amino acid 22, and a lysine residue atβ-globin amino acid 87; (e') an aspartic acid residue at β-globin aminoacid 16, an alanine residue at β-globin amino acid 22, and a lysineresidue at β-globin amino acid 80. Alternatively, the anti-sicklinghuman hemoglobin may include an arginine residue at a-globin amino acid48.

The anti-sickling hemoglobin of the invention is preferably encoded bypurified DNA, for example, purified DNA which includes a hemoglobinsequence encoding any of the above-listed anti-sickling hemoglobins ofthe invention.

Finally, the invention features methods for correcting a sickle defectin a mammal by gene therapy. This method involves administering to themammal a purified nucleic acid encoding a recombinant anti-sicklinghemoglobin of the invention, the anti-sickling hemoglobin nucleic acidbeing positioned for expression in the mammal. In preferred methods, theanti-sickling hemoglobin-encoding nucleic acid is delivered to themammal as part of a viral vector and is delivered to the mammal's bonemarrow. Preferred viral vectors include, but are not limited toretroviral and adeno-associated viral vectors, and any modified versionsof these vectors.

The term "recombinant", as used herein, means expressed from an isolatedor purified DNA molecule. The recombinant anti-sickling hemoglobinsdescribed herein are produced by directed modifications (e.g., by sitedirected or PCR mutagenesis) of such an isolated DNA molecule.

The term "purified DNA", as used herein, means DNA that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (i.e., one at the 5' end and one at the 3'end) in the naturally occurring genome of the organism from which theDNA was derived. The term therefore includes, for example, a recombinantDNA molecule which is incorporated into a vector, e.g., an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA or agenomic fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes a recombinant DNA whichis a part of a hybrid gene encoding additional polypeptide sequences.

The term "human hemoglobin", as used herein, means a molecule whoseamino acid sequence at least in part corresponds to the amino acidsequence of a naturally-occurring human hemoglobin molecule, whethermutated or wild-type.

The term "anti-sickling", as used herein, means capable of interferingwith the aggregation of hemoglobin into 14-stranded hemoglobin moleculescharacteristic of Hb S hemoglobin and resulting in sickle cell anemia(as described herein). Preferably, the anti-sickling molecules of theinvention have approximately the same anti-sickling properties as fetalHb (i.e., α₂ γ₂) hemoglobin (e.g., as measured by in vitro solubilityassays, e.g., the assay of Benesch et al., J. Biol. Chem. 254:8169,1979).

The term "Hb S hemoglobin" as used herein means that hemoglobin whichaggregates into 14-stranded fibers at high intracellular concentrationsand low partial pressure; such Hb S hemoglobin has an A to Ttransversion in the 6th codon of the human β-globin gene.

The term "positioned for expression" means that the DNA molecule ispositioned adjacent to DNA sequences which direct transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,anti-sickling hemoglobin).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated by reference. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methodologies well known toone of ordinary skill in the art. The materials, methods, and examplesare illustrative only, and not limiting.

The invention described herein provides a straightforward approach forthe correction of a sickle cell anemia defect, and thus has importanttherapeutic value. Other features and advantages of the invention willbe apparent from the following description of the preferred embodimentsthereof, and from the claims.

DETAILED DESCRIPTION

The drawings are first described.

DRAWINGS

FIG. 1A is an electron micrograph of an Hb S fiber and a schematicrepresentation of an Hb S fiber.

FIG. 1B is an illustration of the structure of the Hb S fiber. Eachcircle represents a Hb S tetramer. The fiber is composed of seven pairsof double stranded polymers; two double stranded polymers (4 strands)form the inner core and five double stranded polymers (10 strands) formthe outer sheath. Two types of contacts occur between Hb S tetramersincorporated into fibers. Contacts along the long axis of the fiber aretermed axial contacts, while contacts along the sides of tetramers arelateral contacts. The β6 valine plays a critical role in the lateralcontact by interacting with the hydrophobic residues β85 phenylalanineand β88 leucine. An important axial contact is the interaction of theβ22 glutamic acid with the imidazole group of the β20 histidine on anadjacent tetramer.

FIG. 2 is an illustration of a lateral contact in the double stranded HbS polymer. This contact forms when the β6 valine of sickle hemoglobininteracts with a hydrophobic pocket on an adjacent tetramer. Thishydrophobic pocket consists primarily of the residues β85 phenylalanine(phe) and β88 leucine (leu). These two residues are essential forcorrect positioning of the heme moiety and cannot be mutated. However, athreonine (thr) residue at position 87 can be replaced by a glutamine(gln), shown in red. The longer side chain of the glutamine prevents theβ6 valine from interacting with the hydrophobic pocket.

FIG. 3 is an illustration of an axial contact in the double stranded HbS polymer. The side chains of the amino acids B17 lysine (lys), β19asparagine (asn), and β22 glutamic acid (glu) project to form a surfacewhich stabilizes the axial contact. In several of the anti-sicklinghemoglobins, the β22 glutamic acid is replaced by an alanine residue(ala). This residue fails to interact with the positively-chargedhistidine from the neighboring tetramer and thus disrupts the axialcontact. The new alanine residue is shown in light blue.

FIG. 4 is an illustration of 5' HS 1-5 αl and HS 1-5 β^(AS) constructs.One hundred kilobases of the human β-globin locus and 35 kilobases ofthe human α-globin locus are illustrated. Cosmids containing HS 1-5 αland HS 1-5 β^(AS) were constructed by fusing either the α1 gene or arecombinant anti-sickling β-globin gene downstream of the β-globin locuscontrol region (LCR). The 26 kb inserts were purified from vectorsequences, mixed at a 1:1 molar ratio (final DNA concentration was 2ng/μl), and co-injected into fertilized mouse eggs. Transgenic linesdisplaying high-level, balanced expression of the transgenes wereestablished.

FIGS. 5A-5D are graphs of chromatographs of hemolysates andHPLC-purified anti-sickling hemoglobins. Fig. 5A is a graph of achromatograph of hemolysate obtained from transgenic mice expressing HbAS1. Hemoglobins were separated by non-denaturing HPLC. Twenty eightpercent of the hemoglobin in erythrocytes of these animals isrecombinant human αβ^(AS1). FIG. 5B is a graph of denaturing HPLCanalysis of αβ^(AS1) purified from the hemolysate shown in FIG. 5A.Purification was performed by preparative isoelectric focusing (IEF).Approximately 10% of the β-globin chains were of murine origin. FIG. 5Cis a graph of a chromatograph of hemolysate obtained from transgenicmice expressing Hb AS2. Hemoglobins were separated by non-denaturingHPLC. Eighteen percent of the hemoglobin in the erythrocytes of theseanimals is recombinant human αβ^(AS2). FIG. 5D is a graph of denaturingHPLC analysis of αβ^(AS2) purified from the hemolysate shown in FIG. 5C.Purification was performed by preparative IEF. This hemoglobin lacks anycontaminating murine globins. Hemoglobins purified by preparative IEFwere used in all subsequent experiments.

FIGS. 6A-6D are oxygen equilibrium curves (OECs) for purified humananti-sickling hemoglobins. FIG. 6A is an OEC curve for Hb AS1 at pH 7.0in 0.1M potassium phosphate (KPO₄) buffer at 20° C. FIG. 6B is an OECcurve for Hb AS1 under the same conditions as those described in FIG.6A, with the addition of 2 mM 2,3-diphosphoglycerate (DPG). FIG. 6C isan OEC curve for Hb AS2 at pH 7.0 in 0.1M KPO₄ buffer at 20° C. FIG. 6Dis an OEC curve for Hb AS2 under the same conditions as those describedin FIG. 6C with the addition of 2 mM 2,3-DPG.

FIGS. 7A-7B are graphs showing polymerization delay times fordeoxygenated mixtures of human hemoglobins. FIG. 7A shows delay timesfor hemoglobin mixtures containing 100% Hb S or 75% Hb S, together with25% Hb A, Hb AS1, Hb AS2, or Hb F. Curves were determined at ahemoglobin concentration of 60 mg/dl using the temperature jump method(Adachi et al., J. Biol. Chem. 254:7765, 1979). The delay time is anindication of the ability of a hemoglobin to disrupt the polymerizationof Hb S. The delay time of Hb AS1 is between that of Hb A and Hb F,while the delay time of Hb AS2 is similar to that of Hb F at thishemoglobin concentration. FIG. 7B shows delay time vs. hemoglobinconcentration. The progression of the plots from left to rightdemonstrates the increased Hb concentrations which are required forpolymerization to occur in the presence of the various non-Shemoglobins. The delay time plots for Hb AS2 and Hb F overlap,indicating that the anti-polymerization activities of Hb AS2 and Hb Fare virtually identical.

FIG. 8 is an illustration of a retroviral vector useful for theproduction of anti-sickling hemoglobin.

Anti-Sickling β-globin Genes Designed to Inhibit Hb S Polymerization

Recombinant hemoglobins of the invention which contain anti-sicklingmutations can be used to inhibit Hb S polymerization, and thusfacilitate therapies for sickle cell anemia. In particular, the glutamicacid to valine change at the 6th position of the β^(S) polypeptidecreates a non-polar surface that readily interacts with a naturalhydrophobic pocket in the β chain of a second tetramer. This naturalpocket is formed primarily by a phenylalanine (phe) at position 85 and aleucine (leu) at position 88. This interaction leads to the formation ofthe complex 14-stranded fibers described above, and illustrated in FIGS.1A-1B (Bunn et al., Hemoglobin: Molecular, Genetic, and ClinicalAspects, 1986, W. B. Saunders, Philadelphia).

The structure of the fiber that forms in sickle erythrocytes was derivedfrom X-ray diffraction studies of Hb S crystals (Edelstein, J. Mol.Biol. 150:557, 1981). Hb S tetramers are composed of two α-globinsubunits (α₂) and two β^(S) -globin subunits (β^(S) ₂), and formcharacteristic double stranded fibers. Interactions along the long axisof the fiber are termed axial contacts, while interactions along thesides of tetramers are lateral contacts (FIG. 1B; Bunn et al.,Hemoglobin: Molecular, Genetic, and Clinical Aspects. (W. B. Saunders,Philadelphia, 1986)). The β6 valine plays a critical role in the lateralcontact by interacting with the hydrophobic residues β85 phenylalanineand β88 leucine (FIG. 2). Accordingly, to interfere with detrimental HbS polymerization, this interaction and, thus, hydrophobic pocketformation should be disrupted. Because Hb A (α₂ β₂) has these samehydrophobic residues and is readily incorporated into sickle fibers, itcannot be used for this purpose. Moreover, although disruption of thispocket represents the best approach for inhibiting Hb S polymerization,certain strategies have detrimental side effects. For example, althoughamino acid substitutions at β85 phe and β88 leu would interfere withpocket formation, these amino acids are also important for correctpositioning of the heme moiety, and cannot be mutated without severelyaltering oxygen affinity (Dickerson et al., Hemoglobin: Structure,Function, Evolution, and Pathology. (Benjamin/Cummings, Menlo Park,Calif., 1983)).

A better approach for inhibiting Hb S polymerization is depicted in FIG.2 which shows a computer model of a β87 threonine (thr) to glutamine(gln) substitution that disrupts the hydrophobic pocket, withoutinhibiting β-globin function (Perutz et al., Nature 219:902-909, 1968;Computer graphics generated using an Evans and Sutherland PS300 systemrunning the package FRODO (Jones, Meth. Enz. 115:157, 1985)). The longside chain of glutamine prevents the β6 Val from interacting with thehydrophobic pocket. Human γ- and δ-globin polypeptides both have such aglutamine at position 87, and both Hb F (α₂ γ₂) and Hb A2 (α₂δ₂) havepotent anti-sickling activity (Nagel et al., Proc. Natl. Acad. Sci., USA76(2):670-672, 1979). Another naturally occurring human hemoglobin,designated Hb D Ibadan, also has anti-sickling activity (Watson-Williamset al., Nature 205:1273, 1965). This hemoglobin has a lysine at position87 and its long side chain also projects across the hydrophobic pocketand inhibits interactions with the β6 Val.

Preferably, to produce a recombinant anti-sickling hemoglobin, themutations described above (which interfere with a major lateral contact)are combined with a second mutation which interferes with an axialcontact. One such axial contact-disrupting mutation is shown in FIG. 3.The side chains of the amino acids lysine-17 (lys), asparagine-19 (asn),and glutamic acid-22 (glu) project to form a surface which stabilizesthe axial contact with another sickle hemoglobin tetramer (Dickerson etal., Hemoglobin: Structure, Function, Evolution, and Pathology.(Benjamin/Cummings, Menlo Park, Calif., 1983)). Although mutations atresidues 17 or 19 are detrimental, amino acid 22 can be mutated fromglutamic acid to alanine (ala) without an alteration in hemoglobinfunction (Bowman et al., Biochemical and Biophysical ResearchCommunications 26(4):466-470, 1967; Bunn et al., Hemoglobin: Molecular,Genetic, and Clinical Aspects. (W. B. Saunders, Philadelphia, 1986)).The negative charge of the glutamic acid side chain at this positionplays a key role in stabilizing the axial contact because it interactswith the positively charged imidazole group of a histidine at position20 in the α chain of the neighboring tetramer. The shorter nonpolaralanine side chain fails to stabilize this interaction, thus disruptingthe axial contacts between sickle hemoglobin tetramers. The substitutedalanine residue is shown in light blue in FIG. 3. Hb AS2 contains aglutamine at position 87 together with an alanine at position 22. Hb AS1has the same β22 alanine and asparagine at β80 is replaced by lysine.This β80 lysine significantly inhibits sickling when present as a singlesite mutation in Hb A (Nagel et al., Nature 283:832, 1980). Thefollowing 27-mer oligos were used for mutagenesis at the indicated aminoacids in β-globin: β22, GTGAACGTGGATGCCGTTGGTGGTGAG (SEQ ID NO: 1); β80,GCTCACCTGGACAAGCTCAAGGGCACC (SEQ ID NO: 2); β87,GGCACCTTTGCCCAGCTGAGTGAGCTG (SEQ ID NO: 3).

Another anti-sickling mutation in the human β-globin gene useful in theinvention is the Hb G Szuhu mutation, a β80 asn to lys mutation whichhas significant anti-sickling activity (Nagel et al., Proc. Natl. Acad.Sci. USA 76(2):670-672, 1979), but which does not impair hemoglobinfunction (Kaufman et al., Human Heredity 25:60-68, 1975). This mutationis preferably combined with the β22 glu to ala mutation described above.

Alternatively, an α-globin mutation may be utilized to inhibit Hb Spolymerization. One example of such an α-globin mutation is provided bythe hemoglobin designated Hb Montgomery (Brimhall et al., Biochim.Biophys. Acta. 379(1):28-32, 1975), which contains an α48 leucine toarginine mutation. The 54 year old patient from which this mutation wasisolated was homozygous for β^(S), but had no history of painful sicklecell crises, jaundice, leg ulcers, or stroke, and was only mildly anemic(Prchal et al., Am. J. Med. 86(2):232-236, 1989).

Anti-sickling hemoglobin AS3 combines the mutations at β22 and β87,which are present in anti-sickling hemoglobin AS2, with an additionalmutation which lowers the oxygen affinity of the recombinant hemoglobin.The goal is to produce an anti-sickling hemoglobin which delivers oxygento tissues prior to sickle hemoglobin (Hb S). We have termed thisconcept "preferential deoxygenation." If the anti-sickling hemoglobindelivers oxygen preferentially, Hb S will remain oxygenated and,therefore, will not polymerize.

The mutation which was selected to lower the oxygen affinity of theanti-sickling hemoglobin is a change from asparagine to lysine atposition 108 of the β-globin chain. This is the mutation which ispresent in the naturally-occurring Hb Presbyterian (Moo-Penn et al.,FEBS Letters 92:53-56, 1978). Hb AS3 has the following three mutations:(1) β22 glutamic acid to alanine, (2) β87 threonine to glutamine, and(3) β108 asparagine to lysine.

Two additional anti-sickling hemoglobins, AS4 and AS5, have been madewhich combine the mutations present in Hb AS2 at β22 and β87, withadditional mutations which cause the β-globin subunit to become morenegatively charged. In red blood cells, surface charge is a keydeterminant of the ability of α-globin and β-globin monomers toassociate with each other to form dimers (Bunn, Blood 69:1-6, 1987). Thealpha subunit is somewhat positively-charged, while the beta subunit issomewhat negatively-charged. By increasing the negative charge on theβ-globin subunit, it is possible to increase its affinity for theα-globin subunit. Introduction of an additional negative charge in theanti-sickling hemoglobin will provide β^(AS) polypeptides with acompetitive advantage for interacting with α-globin polypeptides.Consequently, α₂ β^(AS) ₂ tetramers will form more efficiently than α₂β^(S) ₂ tetramers.

Anti-sickling hemoglobins Hbs AS4 and AS5 combine the mutations presentin AS2 with a mutation which increases the negative charge on theβ-globin subunit. One mutation which increases the negative charge onthe β-globin subunit but which does not affect the normal functioning ofthe hemoglobin molecule is a change from lysine to glutamic acid atposition 95. This mutation occurs naturally and is known as HbN-Baltimore. The resulting change in charge is -2, since apositively-charged lysine is replaced by a negatively-charged glutamicacid. This change in charge also allows Hb AS4 and Hb S to bedistinguished by isoelectric focusing. Hb AS4 has the following threemutations: (1) β22 glutamic acid to alanine, (2) β87 threonine toglutamine, and (3) β95 lysine to glutamic acid.

An additional mutation which occurs naturally and which is known toincrease the ability of the β-globin subunit to compete for the α-globinsubunit is known as Hb J-Baltimore. This mutation consists of a changefrom glycine to aspartic acid at position 16 of the β-globin subunit.While this mutation adds only one additional negative charge to theβ-globin chain (compared to the two negative charges added by theN-Baltimore mutation described above), the location of the negativecharge is significant. In fact, Hb J-Baltimore competes even moreeffectively than Hb N-Baltimore for the α-globin subunit. Hb AS5 has thefollowing three mutations: (1) β16 glycine to aspartic acid, (2) β22glutamic acid to alanine, and (3) β87 threonine to glutamine.

The invention includes anti-sickling hemoglobins that contain anycombinations of the individual mutations described above. For example,the β108, β95, and β16 mutations may occur either alone, in combinationwith the β22 mutation, or in combination with the β22 mutation andeither the β80 or either of the above-described β87 mutations.

Mutagenesis of Human α- and β-globin Genes

Mutations may be introduced into the normal human α-and β-globin genesby site-directed mutagenesis. For example, a 3.8 kb BglII-EcoRI fragmentcontaining the human α-globin gene or a 4.1 kb HpaI-XbaI fragmentcontaining the human β-globin gene may be cloned into the pSELECTplasmid (Lewis et al., Nucl. Acids. Res. 18:3439-3443, 1990; pSELECT isavailable from the American Type Culture Collection, Rockville, Md.,ATCC# 68196) using standard methods (see e.g., Maniatis et al., 1989,Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Oligonucleotide mutagenesis is performed,e.g., as described by Lewis et al. (Nucl. Acids. Res. 18:3439-3443,1990). In this procedure, an oligonucleotide which corrects a mutationin the ampicillin resistance gene in the pSELECT plasmid is usedsimultaneously with one or more oligonucleotides designed to createmutations in the globin gene insert.

Briefly, E. coli (JM109; ATCC# 53323) containing the pSELECT plasmidwith globin gene inserts are infected with helper phage (M13K07). Aftergrowing the culture overnight (about 12-16 hours), phage obtained fromthe supernatant are extracted with phenol:chloroform, andsingle-stranded DNA is isolated by standard methods. Oligonucleotidescontaining each of the mutations are annealed to single-stranded DNAtogether with the wild-type ampicillin oligonucleotide, and theseprimers are extended with Klenow for about 90 minutes at 37° C.Double-stranded DNA is transformed into E. coli (BMH 71-18 muts), andthe culture is grown overnight in L-broth containing 75 μg/mlampicillin. DNA obtained from rapid lysis preparations of these culturesis transfected into E. coli (JM109), and colonies are selected onampicillin plates (75 μg/ml). Double-stranded DNA obtained from rapidlysis preparations of these colonies is sequenced (Sanger et al., Proc.Natl. Acad. Sci. USA 74:5463-5467, 1977) using oligonucleotide primerslocated upstream of the mutagenic oligonucleotides. Mutants are clearlyidentified by comparison to wild-type sequence.

Construction of Cosmid Clones

The DNA constructs used to produce transgenic animals that synthesizehigh levels of anti-sickling hemoglobins are illustrated in FIG. 4.Constructs used for microinjection are as described by Behringer et al.(Science 245:971, 1989), except that the gene for sickle hemoglobin isreplaced with genes encoding anti-sickling hemoglobins. Mutations areintroduced into the human β-globin gene by site-specific mutagenesis, asdescribed above, and the mutant sequences are inserted downstream of a22 kb DNA fragment containing the DNAse hypersensitive sites 1-5 (5' HS1-5) of the β-globin LCR (Lewis et al., Nucleic Acids Res. 18:3439,1990), as described in further detail below.

In order to construct cosmid clones containing mutant α- and β-globingenes, the mutant genes are excised from pSELECT plasmids and subclonedinto "right arm" plasmids containing a Cos site. Specifically, a 1.2 kbNcoI-XbaI fragment from the α-globin pSELECT plasmids and a 1.4 kbClaI-BamHI fragment from the β-globin pSELECT plasmids are inserted intoright arm plasmids in place of the corresponding α- and β-globin genewild-type fragments. The α-globin right arm plasmids are digested withClaI and MluI, and 4.8 kb fragments containing mutant α-globin geneswhich are linked to Cos sites are purified by agarose gelelectrophoresis. The β-globin right arm plasmids are digested with ClaIand HindIII, and 6.5 kb fragments containing mutant β-globin genes whichare linked to Cos sites are purified similarly. Cosmids containing thesefragments are constructed in four way ligations (Ryan et al., Genes Dev.3:314-323, 1989). The left arms are 9.0 kb MluI-SalI fragments obtainedfrom the cosmid vector pCV001 (Lau et al., Proc. Natl. Acad. Sci. U.S.A.80:5225-5229, 1983). This fragment contains a Cos site, an ampicillinresistance gene, a ColE1 origin and the SVneo gene. The two internalfragments are a 10.7 kb SalI-KpnI fragment containing DNase Isuper-hypersensitive (HS) sites V, IV and III, and a 10.9 kb KpnI-ClaIfragment containing HS II and I. The four fragments are ligated togetherin a 2:1:1:2 molar ratio of vector arms to inserts and packaged(Packagene; Promega, Madison, Wis.). E. coli ED8767 is infected with thepackaged cosmids and is plated onto ampicillin plates. Large scalecultures of ampicillin resistant colonies are grown, and cosmids areprepared by standard procedures.

Transgenic animal assays--Characterization of anti-sickling hemoglobins

The effects of anti-sickling hemoglobin can be analyzed using transgenicanimals. Cosmid DNA is prepared by standard procedures. HS I-V α and HSI-V β cosmids containing the mutations described above are eitherinjected directly into fertilized mouse eggs, or the constructs aredigested with SalI and insert DNA is separated from plasmid DNA byagarose gel electrophoresis prior to injection. The injected eggs andtransferred to pseudopregnant foster mothers (Brinster et al., Proc.Natl. Acad. Sci. USA 82:4438-4442, 1985), and transgenic progeny areidentified by Southern blot hybridization of tail DNA. Similarly, largeanimal eggs can be injected with the same constructs and transferred tofoster mothers as described by Pursel et al. (Science 244:1281-1288,1989). Typically, human α- and β-globin genes are cloned into expressionvectors designed to direct high levels of α- and β-globin synthesis inerythroid cells of transgenic animals. These constructs are co-injectedinto fertilized mouse eggs and expression is analyzed in transgenicanimals that develop.

Blood collected from transgenic animals is washed with saline, andhemolysates prepared as described by Ryan et al. (Science 245:971-973,1990). Hemoglobin is analyzed on isoelectric focusing (IEF) gels (Ryanet al., Science 245:971-973, 1990) to demonstrate that a complete humanhemoglobin is formed in adult erythrocytes, and to identify transgenicanimals which synthesize high levels of human hemoglobin (Ryan et al.,Science 247:566, 1990; Behringer et al., Science 245:971, 1989). Humanhemoglobin bands are excised from IEF gels and analyzed on ureacellulose acetate strips to demonstrate that the human hemoglobin bandis composed of human α- and β-globin polypeptides. It is noted that ifhuman hemoglobin is difficult to separate from endogenous hemoglobins,mutations that increase or decrease the isoelectric point (pI) of humanhemoglobin can be introduced into the α- and β-globin genes. Increasesin pI are accomplished by introducing basic (positively charged) aminoacids into the protein, while decreases in pI are accomplished byintroducing acidic (negatively charged) amino acids. These charged aminoacids are introduced at positions which do not disturb the structure orfunction of the protein. Oxygen equilibrium curves (OECs) of humanhemoglobin purified from the transgenic mice are determined as describedby Ryan et al. (Science 247:566-568, 1990).

The anti-sickling properties of the AS hemoglobins (purified fromerythrocytes of the above-described transgenic animals) can bequantitated by in vitro solubility assays as described, e.g., by Beneschet al. (J. Biol. Chem. 254:8169, 1979). Briefly, the anti-sicklinghemoglobin is mixed with Hb S. The solution is cooled to 0° C.,deoxygenated, and then incubated at 30° C. for 2 to 3 hours. Insolublepolymers are pelleted by ultracentrifugation, and the concentration ofhemoglobin in the supernatant is determined spectrophotometrically. Thesolubility of mutant hemoglobin/Hb S mixtures is compared with Hb A/Hb Sand Hb F/Hb S solutions.

Retroviral Vectors Designed To Correct the Sickle Defect

The anti-sickling hemoglobin genes described herein may be used tocorrect a sickling defect by gene therapy. Such techniques are firsttested in an animal model, for example, mice, but similar techniques maybe used to treat other mammals, including humans. A description of aretroviral vector useful for transferring anti-sickling hemoglobin genesto a mammal now follows.

As a first step toward gene therapy, the anti-sickling β-globin genesdescribed above are preferably inserted into a retroviral vector such asthat illustrated in FIG. 4. This vector is of a small size to optimizethe viral titers obtained. To construct such a small vector, a minimumHS 2 region (Caterina et. al., Proc. Natl. Acad. Sci. USA 88:1626-1630,1991) of the globin LCR is inserted upstream of the β-globin gene; this1.1 kb KpnI-XbaI fragment containing HS 2 retains sequences required forboth enhancer and domain opening activity, facilitating high levelexpression of downstream β-globin genes with minimum size. In addition,the vectors include mini-globin genes which contain only 200 bp of 5'flanking sequence and 150 bp of 3' flanking sequence, and only 100 bp ofIVS 2. The mini-globin genes, although small, contain all of thesequences necessary for high level expression, including the TATA (-35),CCAAT (-70) and CACCC (-100) boxes (Antoniou et al., Genes Dev.4:1007-1013, 1990; Antoniou et al., Genes Dev. 4:1007-1013, 1990). Theyalso contain all sequences required for correct splicing of the β-globinsecond intron, including the splice donor, splice acceptor, and branchpoint sequences.

Another feature of these constructs is the use of a deleted SV-Neoregion (SN) of LXSN (Miller et al., Biotechniques 7(9):980-990, 1989);this deletion removes 1.5 kb of DNA and significantly reduces the sizeof the construct. Although these viruses cannot be easily titered byconventional means, viral titers can be estimated by Southern blothybridization of NIH 3T3 cells that are infected with supernatants frompackaging cells lines. Briefly, to carry out such an assay, theconstructs described above are co-transfected with an SV-Neo plasmidinto an ecotropic and/or amphotropic packaging cell line (for example,E86 and PA317) (Markowitz et al., Virology 167(2):400-406, 1988;Markowitz et al., J. Virol. 62(4):1120-1124, 1988; Miller et al., Mol.Cell Biol. 6:2895-2902, 1986), and colonies of G418 resistant cells areisolated. Undiluted and serially diluted supernatants from thesecolonies are used to infect NIH 3T3 cells; a high titer LXSN virus isused as a control. Southern blot hybridizations with an LX specificprobe identify supernatants that efficiently transduce intact copies ofthe retrovirus to 90-100% of the cultured cells.

If desired, before transfecting retroviral DNAs into packaging celllines, the constructs may be tested for expression in 16 day fetal liverof transgenic mice as described by Ryan et al. (Genes Dev. 3:314-323,1989). Constructs that are expressed at a high level are used to producevirus for bone marrow infections.

When a packaging cell line that produces high titer virus (preferably,10⁶ /ml) is obtained, bone marrow from Hb S mice is infected; productionof such Hb S mice is carried out as described above using mutant Hb Shemoglobin transgenes (see, e.g., Ryan et. al., Science 247:566-568,1990). To facilitate infection, bone marrow from Hb S mice areco-cultured with the packaging line in the presence of IL-3 and IL-6(Bodine et al., Proc. Natl. Acad. Sci. USA 86(22):8897-901, 1989). After48 hours, cells are injected via the tail vein into recipient Hb Sanimals that have been lethally irradiated. After a one month recoveryperiod, small aliquots of blood are removed and hemoglobins are analyzedon native IEF gels and denaturing cellulose acetate strips (see, e.g.,Behringer et. al., Science 245:971-973, 1989. Preparative IEF wasperformed on 4% acrylamide gels with 2% Pharmalyte pH 6.7 to 7.7. Bandsof hemoglobin were sliced from the gel and eluted in 0.1M potassiumphosphate buffer, pH 7.0). When animals that express human β-globin atapproximately 20% of total β-globin are obtained, solubility assays areperformed to quantitate anti-sickling activity. Also, erythrocytes fromthese animals are deoxygenated and examined for sickled forms. Resultsare compared to control animals that have been transplanted with Hb Smarrow infected with LXSN virus only.

Mutant hemoglobins shown to inhibit sickling in Hb S mice are thenincluded in the appropriate mammalian retroviral vector and introducedinto a mammal of choice, generally as described above. Retroviralvectors, or other viral vectors with the appropriate tropisms for bloodcells, may be used as gene transfer delivery systems for theanti-sickling hemoglobin gene. Numerous vectors useful for this purposeare generally known and have been described (see for example, Miller,Human Gene Therapy 1:5-14, 1990; Friedman, Science 244:1275-1281, 1989;Anderson, Science 256:808-813, 1992; Eglitis et al., BioTechniques6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology1:55-61, 1990; Cornetta et al., Nucleic Acid Research and MolecularBiology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen,Blood Cells 17:407-416, 1991; and Miller et al., Biotechniques7:980-990, 1989). Retroviral vectors are particularly well developed andhave been used in a clinical setting (see, for example, Rosenberg etal., N. Engl. J. Med. 323:370, 1990). Preferably, the anti-sicklinghemoglobin genes are introduced by retroviral transfer into a sample ofa patient's bone marrow stem cells (also as described herein and inMiller, 1990, supra; Friedman, 1989, supra; Anderson, 1992, supra;Eglitis et al., 1988, supra; Tolstoshev et al., 1990, supra; Cornetta,1987, supra; Anderson, 1984, supra; Moen, 1991, supra; Miller et al.,1989, supra; and, Rosenberg et al., 1990, supra).

Example: Characterization of anti-sickling hemoglobins AS1 and AS2produced in transgenic mice

Transgenic lines expressing AS1 or AS2 were established, and hemolysatesobtained from several animals were analyzed by anion exchange highperformance liquid chromatography (HPLC) to quantitate the amounts ofhuman, mouse, and hybrid hemoglobins (Ip et al., Anal. Biochem. 156:348,1986; Hemoglobin tetramers were separated by anion exchange HPLCutilizing a Synchropak AN 300 (4.6 mm×25 mm) column (SynChrom,Lafayette, Ind.)). FIGS. 5A and 5C show that 28% of total hemoglobin wasHb AS1 in one αβ^(AS1) transgenic line, and 18% of total hemoglobin wasHb AS2 in one αβ^(AS2) transgenic line. Hemoglobins AS1 and AS2 wereisolated by preparative IEF (Behringer et al., Science 245:971, 1989)and the purity of the human hemoglobins was assessed by denaturingreverse phase (HPLC) which separates the α- and β-globin subunits(Adachi et al., J. Chromat. 419:303, 1987. Mouse and human globins wereseparated by RP-HPLC using a Dionex Series 4500i HPLC system (Sunnyvale,Calif.). Approximately 25-30 μg of hemoglobin was injected into a VydacC4 reversed phase column (4.6 mm×250 mm; Hibernia, Calif.) and elutedwith a linear gradient of acetonitrile and 0.3% trifluoroacetic acid asdescribed in Shelton et al., J. Liq. Chrom. 7:1969, 1977). FIGS. 5B and5D show that Hb AS1 was approximately 90% pure, while Hb AS2 waspurified to homogeneity.

The oxygen equilibrium curves (OEC) for purified Hb AS1 and Hb AS2 areillustrated in FIGS. 6A and 6C (Asakura et al. in Oxygen Transport inRed Blood Cells, C. Nicolau Ed. (Pergamin, N.Y., 1986). Oxygenequilibrium curves were measured with a Hemox Analyzer (TCS,Southampton, Pa.). The OEC were determined in 0.1M potassium phosphatebuffer, pH 7.0 at 20° C.). These sigmoidally shaped curves demonstratethe normal cooperativity of oxygen binding (FIGS. 6A and 6C). The P₅₀value, which measures the partial pressure of oxygen at which hemoglobinis half-saturated, was determined for Hb AS1 and Hb AS2 and comparedwith Hb A and Hb F (Table 1). The P₅₀ for Hb AS1 is slightly elevated,but within the normal range, and this hemoglobin responds normally tothe allosteric effector 2,3-diphosphoglycerate (2,3-DPG); that is,oxygen affinity is decreased in the presence of 2 mM 2,3-DPG (FIG. 6B).The P₅₀ for Hb AS2 is slightly lower than normal (6.7 mm Hg) but 2,3-DPGraises this value to 8.4 mm Hg. The oxygen affinity of Hb AS2 isfunctionally equivalent to Hb F in the presence of 2 mM 2,3-DPG (FIG.6D) and, therefore, Hb AS2 should adequately bind and deliver oxygen invivo.

                  TABLE 1    ______________________________________    P.sub.50 values for recombinant and naturally-occurring    human hemoglobins                P.sub.50 (mm Hg)    Sample        without DPG                            with DPG    ______________________________________    Hb AS1        10.5      15.0    Hb AS2        6.7       8.4    Hb A          8.7       13.3    Hb F          8.8       10.0    ______________________________________

Anti-sickling Properties of AS1 and AS2 hemoglobins

Hb S (100%) or mixtures of Hb S (75%) and Hb A, AS1, AS2, or F (25%)were deoxygenated and polymerization as a function of time was measuredspectrophotometrically as the temperature of the hemoglobin solution wasraised from 0° C. to 30° C. (Adachi et al., J. Biol. Chem. 254:7765,1979; Adachi et al., J. Biol. Chem. 255:7595, 1980; Kinetics ofpolymerization were determined in 1.8M potassium phosphate buffer.Polymerization was initiated using the temperature jump method in whichthe temperature of deoxygenated hemoglobin solutions is rapidly changedfrom 0° C. to 30° C. and the time course of aggregation is monitoredturbidimetrically at 700 nm). FIG. 7A shows that Hb S polymerizesrelatively rapidly and that Hb A, AS1, AS2, and F delay Hb Spolymerization to different extents. Hb AS1 inhibits Hb A polymerizationmore efficiently than Hb A; however, Hb AS1 inhibits much lesseffectively than Hb F which is known to inhibit sickling in vivo at a3:1 ratio (Noguchi et al., New Eng. J. Med. 318:96, 1988). Finally, HbAS2 inhibits Hb S polymerization at approximately the same level as HbF. This result strongly suggests that Hb AS2 will inhibit Hb Spolymerization in vivo if expression of AS2 at a level of 25% of totalhemoglobin can be achieved.

The delay times determined in FIG. 7A were all measured at aconcentration of 60 mg/dl. FIG. 7B shows the results of similarexperiments performed at variable concentrations of total hemoglobin.The ratio of Hb S to Hb A, AS1, AS2 or F in all of these experiments was3:1. In this figure the log of the reciprocal of the delay time and thelog of hemoglobin concentration are plotted. As reported previously(Hofrichter et al., Proc. Natl. Acad. Sci. USA 71:4864, 1974; Wishner etal., J. Mol. Biol. 98:179, 1975; Crepeau et al., Nature 274:616, 1978;Dykes et al., J. Mol. Biol. 130:451, 1979; Padlan et al., J. Biol. Chem.260:8280, 198; and Eaton et al., Blood 70:1245, 1987), an empiricalrelationship between delay time and hemoglobin concentration can bedescribed by the following equation: 1/td=γS^(n), where S= Hb!total/Hb!soluble, and γ is an experimental constant. The n value is related tothe size of nuclei formed during polymerization. The n values of thedata shown in FIG. 7B are between 2 and 3, which agree well with thoseshown previously in high phosphate buffer (Adachi et al., J. Biol. Chem.254:7765, 1979). At higher concentrations of hemoglobin, the delay timesfor Hb AS2 and Hb F overlap, indicating that Hb AS2 and Hb F havevirtually identical anti-polymerization activity.

The results described above demonstrate that the genetic modification oftwo surface amino acids in Hb A produces a unique human hemoglobin (HbAS2) that inhibits Hb S polymerization as effectively as Hb F. Asdiscussed above, the β-globin LCR enhances β-globin gene expression muchmore effectively than γ-globin gene expression in adult erythroid cells.Therefore β^(AS2), which is a β-globin gene with the anti-polymerizationproperties of γ-globin, a useful molecule for future genetic therapy ofsickle cell disease.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    GTGAACGTGGATGCCGTTGGTGGTGAG27    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    GCTCACCTGGACAAGCTCAAGGGCACC27    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    GGCACCTTTGCCCAGCTGAGTGAGCTG27    __________________________________________________________________________

What is claimed is:
 1. A recombinant human β-globin with anti-sickling activity, said recombinant human β-globin having amino acid substitutions in amino acid positions 22 and
 80. 2. The recombinant human β-globin of claim 1, comprising alanine at amino acid position
 22. 3. The recombinant human β-globin of claim 1, comprising lysine at amino acid position
 80. 4. The recombinant human β-globin of claim 1, comprising alanine at amino acid position 22 and lysine at amino acid position
 80. 5. A recombinant human β-globin with anti-sickling activity, said recombinant human β-globin having amino acid substitutions in amino acid positions 22 and
 87. 6. The recombinant human β-globin of claim 5, comprising alanine at amino acid position
 22. 7. The recombinant human β-globin of claim 5, comprising glutamine at amino acid position
 87. 8. The recombinant human β-globin of claim 5, comprising lysine at amino acid position
 87. 9. The recombinant human β-globin of claim 5, comprising alanine at amino acid position 22 and glutamine at amino acid position
 87. 10. The recombinant human β-globin of claim 9, further comprising an amino acid substitution at amino acid position
 16. 11. The recombinant human β-globin of claim 10, comprising aspartic acid at amino acid position
 16. 12. The recombinant human β-globin of claim 5, comprising alanine at amino acid position 22 and lysine at amino acid position
 87. 13. The recombinant human β-globin of claim 12, further comprising an amino acid substitution at amino acid position
 16. 14. The recombinant human β-globin of claim 13, comprising aspartic acid at amino acid position
 16. 15. The recombinant human β-globin of claim 5, further having an amino acid substitution at amino acid position
 16. 16. The recombinant human β-globin of claim 15, comprising aspartic acid at amino acid position
 16. 